Gene panel participating in liver regeneration

ABSTRACT

A gene panel comprising a group of genes of which expression levels change in hepatocytes during liver regeneration as compared with those in a normal state is produced by the following steps: (a) the step of measuring expression levels of various genes in hepatocytes of a model animal in a normal state and expression levels of the genes during liver regeneration; (b) the step of comparing the expression levels, respectively; and (c) the step of identifying a group of genes of which expression levels change during liver regeneration.

TECHNICAL FIELD

The present invention relates to a gene panel comprising a group ofgenes of which expression levels change in hepatocytes during liverregeneration as compared with those in a normal state and a method forproducing the same as well as use of the gene panel. These aspects ofthe present invention are useful in the fields of diagnosis, drug and soforth.

BACKGROUND ART

After partial hepatectomy in liver cancer treatment or transplantationof partially resected liver in liver disease treatment, rapidregeneration of the remaining liver or transplanted liver is important.However, poor liver regeneration is observed in not a few cases.Further, adults often have fatty livers, and fatty livers often lead topoor regeneration, which causes problems after partial hepatectomy.Furthermore, in order to compensate for damaged liver in hepatitis, itis important to accelerate liver regeneration. Delay in the regenerationworsens hepatitis and can lead to fulminant hepatitis, of whichprognosis is poor.

Accordingly, medical care that accelerates liver regeneration is beingrequired, and it can be said that screening for drugs accelerating liverregeneration is important. However, in spite of such requirement, noeffective therapeutic agent that accelerates liver regeneration has beenfound, and no method for efficiently screening for drugs that accelerateliver regeneration has been known.

It is known that, when partial hepatectomy is performed in a rat, liverregeneration is quickly started, and that the hepatocyte population sizecomes back to the size before the hepatectomy in about 1 week. While itis considered that liver regeneration is accelerated by interactions oftwo or more kinds of associated genes, the overall picture of liverregeneration process remains unknown for what kinds of genes function atwhat kinds of timings. Since it is considered that many genes areinvolved in liver regeneration, it is considered that it is important toexamine a large number of important genes involved in liver regenerationto grasp the overall picture of liver regeneration. However, althoughattempts have been made so far to reproduce liver regeneration in aculture system including a combination of genes and proteins consideredto be involved in liver regeneration, no one has succeeded yet. Further,gene screenings utilizing changes in expression observed during liverregeneration as index have been reported (Xu W et al., Biochem. Biophys.Represents. Commun., 278(2): 318-25, 2000; Kar S and Carr B I, Biochem.Biophys. Represents. Commun., 212(1): 21-6, 1995; Mohn K L, et al., Mol.Cell Biol., 11(1): 381-90, 1991; Sobczak J et al., Exp. Cell Res.,169(1): 47-56, 1987; Huber B E et al., Hepatology, 6(2): 209-19, 1986).However, in these screenings, expressions of only a few types of geneshave been examined.

DISCLOSURE OF THE INVENTION

The present invention was accomplished in view of the above, and itsobject is to provide a gene panel comprising genes of which expressionlevels change in hepatocytes during liver regeneration as compared withthose in a normal state and a method for screening for a drug thataccelerates liver regeneration by utilizing the gene panel.

The inventors of the present invention considered that liverregeneration could be accelerated by realizing behaviors of genesinvolved in liver regeneration close to those of their patterns observedwhen liver regeneration was being accelerated. Then, they examinedexpression profiles of various genes during liver regeneration by usingpartially hepatectomized rats to obtain expression information aboutgenes involved in liver regeneration, and thus accomplished the presentinvention.

That is, the present invention provides the followings.

(1) A gene panel comprising names of genes of which expression levelschange in hepatocytes during liver regeneration as compared with thosein a normal state and expression profiles of the genes.

(2) The gene panel according to (1), wherein the changes of the geneexpression levels are changes in the expression levels in a model animalafter partial hepatectomy as compared with the expression levels in anormal state in the model animal.

(3) The gene panel according to (1) or (2), wherein the expressionprofiles include expression profiles over time during liverregeneration.

(4) The gene panel according to any one of (1) to (3), which includessequence information of a group of PCR primers for analyzing theexpression profiles of the genes.

(5) The gene panel according to any one of (2) to (4), wherein the modelanimal is a rat.

(6) The gene panel according to any one of (1) to (5), which includesexpression profiles of at least 6 types of genes among 166 types of thegenes represented by the numbers 1 to 166 in Tables 1, 6 and 7.

(7) The gene panel according to (6), wherein the at least 6 types ofgenes are selected from 151 types of the genes represented by numbers 1to 151 in Tables 1 and 6.

(8) The gene panel according to (6), wherein the at least 6 types ofgenes are selected from 137 types of the genes represented by thenumbers 1 to 137 in Tables 1 and 6.

(9) The gene panel according to any one of (6) to (8), wherein the atleast 6 types of genes are the genes represented by the numbers 5, 17,36, 46, 67 and 68 in Table 1.

(10) A method for producing a gene panel comprising expression profilesof genes of which expression levels change in hepatocytes during liverregeneration as compared with those in a normal state, which comprisesthe steps of:

(a) measuring expression levels of various genes in hepatocytes of amodel animal in a normal state and expression levels of the genes duringliver regeneration;

(b) comparing the expression levels, respectively; and

(c) identifying a group of genes of which expression levels changeduring liver regeneration and producing expression profiles frominformation about gene names and changes in the expression levels.

(11) The method according to (10), wherein, in the step (a), theexpression levels of the gene are analyzed over time during liverregeneration.

(12) The method according to (11), wherein a liver regenerationaccelerating substance is administered before, during or after liverregeneration.

(13) The method according to (12), wherein the liver regenerationaccelerating substance is L-alanine.

(14) The method according to any one of (10) to (13), wherein the geneexpression levels are analyzed by one or more kinds of methods selectedfrom the gene chip method, the ATAC-PCR method and the Taqman PCR (SYBRGreen) method.

(15) The method according to (14), wherein the gene expression levelsare analyzed by the gene chip method and the ATAC-PCR method.

(16) The method according to (14), wherein the gene expression levelsare analyzed by the Taqman PCR (SYBR Green) method.

(17) A method for screening for a drug involved in liver regeneration,which comprises administering a drug to a model animal or liver tissueor cells and profiling expressions of genes constituting the gene panelaccording to (1).

(18) A method for evaluating a condition of liver, which comprisesprofiling expressions of genes constituting the gene panel according to(1) for liver of a subject.

(19) A group of primers used in the method for producing a gene panelaccording to (10), the screening method according to (17) or theevaluation method according to (18), which comprises all or a part ofthe oligonucleotides of SEQ ID NOS: 1 to 127 and 135 to 192.

Hereafter, the present invention will be explained in detail.

<1> Gene Panel of the Present Invention

The gene panel of the present invention is a gene panel comprising namesof genes of which expression levels change in hepatocytes during liverregeneration as compared with those in a normal state and geneexpression profiles of the genes.

The gene panel of the present invention can be produced by the followingsteps:

(a) the step of measuring expression levels of genes in hepatocytes of amodel animal in a normal state and expression levels of the genes duringliver regeneration;

(b) the step of comparing the expression levels, respectively; and

(c) the step of identifying a group of genes of which expression levelschange during liver regeneration and producing expression profiles frominformation about gene names and changes in expression levels.

The liver regeneration refers to a phenomenon that, when a part of cellsin the liver are damaged or lost due to resection or the like, thecondition is repaired and goes back to the original normal livercondition. For example, this phenomenon is observed in the remainingliver or transplanted liver after partial hepatectomy in liver cancertreatment or transplantation of partially resected liver in liverdisease treatment.

The gene expression level is synonymous with expression amount,expression intensity or expression frequency of a gene, and is usuallyanalyzed based on a production amount of a transcription productcorresponding to the gene, activity of its translation product or thelike.

The gene expression level can be measured by methods usually utilized inanalyses of gene expressions. Examples of preferred methods include thegene chip method, the gene microarray method, the gene macroarray methodand so forth. In these methods, gene fragments arrayed and immobilizedon a certain plate (usually, slide glass) are used. This chip ishybridized with fluorescence-labeled mRNA, and the type and amount ofthe mRNA are measured.

Further, as methods preferred in the present invention, the ATAC-PCRmethod and the Taqman PCR (SYBR Green) method can be mentioned. TheATAC-PCR method is a type of the quantitative PCR technique developed byKato et al (Kato, K. et al., Nucl. Acids Res., 25, 4694-4696, 1997) andis characterized in that it enables quantification of expression of atrace amount sample. Further, while conventional quantitative PCRmethods enable quantification of expressions of several tens of types ofgenes at most, the ATAC-PCR method enables quantification of expressionsof 1000 or more types of genes. The Taqman PCR (SYBR Green method) is acommon technique for quantitative RT-PCR (Schmittgen T D, Methods, 25,383-385, 2001) and is characterized in that primers can be readilydesigned. Moreover, its procedure is simple, and expressions of a largenumber of genes can be measured in a short time (40 to 150 genes/2hours).

Besides the above, examples of the gene expression analysis methodinclude the body map method (Gene, 174, 151-158, 1996), serial analysisof gene expression (SAGE) (U.S. Patent Application Nos. 527,154 and544,861, European Patent Publication No. 0761822), micro-analysis ofgene expression (MAGE) (Japanese Patent Laid-open Publication (Kokai)No. 2000-232888) and so forth.

The body map method will be outlined below. A poly-T sequence on avector and a poly-A tail at the 3′ end of mRNA are ligated, and cDNA issynthesized by using the vector poly-T sequence as a primer. Further,the cDNA is digested with a restriction enzyme MboI. Since one MboI siteexists per 300 base pairs on average on cDNA, the cDNA on the vector isdivided into fragments of 300 base pairs in average. At this time, thecDNA fragment closest to the poly-A tail remains as ligated to thevector. The vector having this cDNA fragment is cyclized and introducedinto Escherichia coli to construct a cDNA library. About 1000 clones arearbitrarily selected from the library, and a nucleotide sequence of 300base pairs in average is determined for each clone. Among thesesequences, clones comprising the same sequences are collected, and thetype and the emerging frequency of each sequence are obtained to createa gene expression profile. Homology search (BLAST search) in the databank is performed for each cDNA sequence, and a clone having the samesequence as that of a known gene is given the name of the gene. When thesequence is not registered at the data bank, it is assumed that a genecorresponding to the sequence does not exist.

In order to perform the homology search by the BLAST search, informationof at least 11 base pairs is necessary. There are about one milliontypes of genes having sequences comprising 10 nucleotides. This numberof types is far more than the number of 100,000 types of genes expectedto exist in human. That is, if there is information about 11 base pairs,genes having the sequence can be identified, and the gene expressionprofiles can be analyzed. Therefore, in order to efficiently analyzegene expression profiles by the body map method requiring manysequences, cDNA fragments having about 300 base pairs in the body mapcan be further divided into short fragments having 11 base pairs or more(referred to as “tag”), and these fragments can be further ligated toone another as many kinds of fragments and inserted into a vector toconstruct a ligated tag library. Then, if about 1000 clones arearbitrarily selected as in the body map method to determine the DNAsequences of the ligated tags, it can be expected that information aboutexpressions of more genes can be obtained with the same procedure as inthe body map method. Each tag represents a gene sequence, and theemerging frequency of the tag represents the expression frequency of thegene. Since the length of a DNA sequence that can be determined in onesequencing is usually about 600 base pairs, DNA sequences of about 50tags at most can be determined by one sequencing. That is, geneexpression profile analysis can be performed with about 50 times higherefficiency at most compared with the body map method.

The SAGE method is a method for analyzing gene expression profiles basedon the above concept. The SAGE method is performed as follows. cDNA isprepared by using a poly-T bonded with biotin at the 3′ end as a primerand digested with a restriction enzyme such as MboI (referred to as“anchoring enzyme”) as in the body map, and then a cDNA fragmentincluding the 3′ end bonded with biotin is adsorbed on avidin beads.Then, the beads are divided into two portions, and one of two types oflinkers (A and B) is bonded to the cDNA fragment (about 13 bp) adsorbedon the beads of one of the two portions, respectively. A site for aClass II restriction enzyme such as BsmFI (referred to as “taggingenzyme”) is added to each linker beforehand. The cDNA fragments areexcised from the beads with the tagging enzyme, the digestion sites areblunt-ended, and the tag ligated to the linker A and the tag ligated tothe linker B are ligated to each other. This product is called a ditag.The ditag is amplified by PCR using primers recognizing the linker A andthe linker B. The amplified ditags are ligated to one another as manykinds of fragments and incorporated into a vector and sequenced. About50 tag sequences can be obtained per sequencing. This tag sequenceinformation is collected to obtain the gene expression frequency.

The MAGE method is a modified version of the aforementioned method, andis a method enabling gene expression frequency analysis with highefficiency and precision, in which cDNAs are synthesized from mRNAs byusing a vector primer having a poly-T sequence, the cDNA sequences aretagged on a vector, the obtained tags are ligated via a sequence thatallows identification of the tag end to form a concatemer, and thenucleotide sequence of the concatemer is analyzed.

In the present invention, the gene expression analysis method is notparticularly limited so long as gene expression levels can be analyzed,and any of currently known methods and methods to be developed in futurecan be employed. Among the methods mentioned above, particularlypreferred are the gene chip method, the gene microarray method, theATAC-PCR method and the Taqman PCR (SYBR Green) method.

In the present invention, gene expression levels may be analyzed basedon results obtained by a single kind of method or results obtained bytwo or more kinds of methods. Although the analysis can be performed bya single kind of method to a certain extent, more precise analysis canbe attained by using two or more kinds of methods in combination.Specifically, a correlation coefficient is calculated for resultsobtained by two or more kinds of methods, for example, the gene chipmethod and the ATAC-PCR methods. When the correlation coefficient is acertain value or higher, it is assumed that the expression level of thecorresponding gene has changed.

Various gene chips, gene microarrays and gene macroarrays of human oranimal such as mouse are commercially available, and these may be usedin the present invention.

In the present invention, changes in gene expression levels can beanalyzed by measuring expression levels of various genes in hepatocytesin a normal state and expression levels of these genes during liverregeneration and comparing their expression levels, respectively. Thegene expression levels during liver regeneration are analyzed by, forexample, using a model animal such as a partially hepatectomized rat andmeasuring the gene expression levels in hepatocytes after thehepatectomy. On the other hand, the gene expression levels in a normalstate are typically measured immediately before the hepatectomy. Thegene expression levels are preferably analyzed over time during liverregeneration.

As described above, a group of genes of which expression levels changeduring liver regeneration is identified. Expression profiles of thesegenes are produced by allocating information about the change ofanalyzed expression level to the identified gene, respectively.

Further, in the present invention, as genes of which expression levelschange during liver regeneration as compared with those in a normalstate, that is, candidates of genes considered to be involved in liverregeneration, genes estimated to be involved in the metabolism ofL-alanine can be mentioned.

L-alanine is known to accelerate liver regeneration (Maezono K et al.,Hepatology 24(5), 1996; Japanese Patent Laid-open Publication No.5-229940). Further, elevation of the L-alanine concentration in humanplasma after hepatectomy (Nijveldt R J et al., Liver 21(1): 56-63,2001), accumulation of L-alanine in rat liver tissue after hepatectomy(Brand H S et al., J. Hepatology, 23, 333-340, 1995) and so forth havebeen reported. As data supporting these findings, elevation of mRNAlevel of the amino acid transporter system A (ATA2), which transportsL-alanine, was observed in 70% partially hepatectomized rat as a liverregeneration model, as described later (the examples of the presentspecification). Further, since increase of the L-alanine transportingactivity in a similar liver regeneration model has also been observed(Joan-Vicenc et al., FEBS LETTERS, 329(1,2): 189-193, 1993; Freeman T Let al., BBRC, 278, 729-732, 2000), possibility of increase in uptake ofL-alanine into hepatocytes during liver regeneration is suggested.

Further, the uptake of [³H]thymidine into DNA is decreased when the Alatransport activity of ATA2 is inhibited by a treatment with MeAIB(nonmetabolizable System A-specific substrate,alpha-(methylamino)isobutyric acid) (Freeman T L et al., Hepatology,30(2): 437-444, 1999). Therefore, it is suggested that acceleration ofliver regeneration is suppressed when transport of L-alanine intohepatocytes is inhibited. This also strongly suggests that ATA2 (Ala) isinvolved in acceleration of liver regeneration.

Furthermore, in the field of surgical operations, it has been reportedfrom old days that markedly increased energy metabolism is observed inliver under an invasive condition such as hepatectomy, and amino acidsrepresented by L-alanine are supplied primarily as a result ofdecomposition of myoproteins and used as substrates for gluconeogenesis(Felig P et al., Metabolism, 22, 179-207, 1973) and protein synthesis.As described above, L-alanine is generally considered to supply energythrough gluconeogenesis during liver regeneration. In fact, oxidation ofL-alanine was suppressed in a liver regeneration model rat at 9 or 18hours after hepatectomy, and increase in plasma glucose level,accumulation of liver glycogen and enhanced activities offructose-1,6-diphosphatase and PEPCK have also been reported (Klain G Jet al., J. Nutr. Biochem., 1 (November), 578-584, 1990).

Based on the above findings, it was revealed that L-alanine acceleratedliver regeneration, and it was hypothesized that its action mechanism isbased on the energy supply increased by L-alanine during liverregeneration. Therefore, as shown in the examples described later,expressions of 59 types of rat genes involved in L-alanine metabolismwere analyzed during liver regeneration. As a result, changes inexpression were observed for 17 types of the genes. Accordingly, whenthe gene panel of the present invention is produced, genes involved inthe L-alanine metabolism may be selected prior to the step (a) mentionedabove.

Even for a gene of which expression change is not observed during liverregeneration, expression change may be observed if L-alanine isadministered before, during or after liver regeneration as shown inExample 3. Such a gene may also be included in the gene panel. Further,a substance having an action for accelerating liver regeneration (liverregeneration accelerating substance) other than L-alanine may also beadministered before, during or after liver regeneration. Examples of theliver regeneration accelerating substance include a mixture of liverextract and flavin adenine dinucleotide (15 μl of liver extract, 10 mgof FAD per 1 ml), liver hydrolysate, saiko-keishito, shosaikoto,diisopropylamine dichloroacetate, placenta hydrolysate, valine (WO0113912), vascular endothelial growth factor/vascular permeabilityfactor (VEGF/VPF) (WO 0132213), macrolide compounds (JP 3240728), TCF II(JP 10194986), TGF-β release/activation/synthesis inhibitor (JP8333249),glucose-1-phosphate, glucose-6-phosphate (JP2202821), desialatedorosomucoid (JP 6122025) and so forth.

The gene panel of the present invention includes at least names ofvarious genes identified as described above and expression profiles ofthe respective genes. As for the name of genes, the nomenclature for thegenes is not particularly limited so long as the genes can bedistinguished from one another. Typically, names of products encoded bythe genes, accession numbers or gene names used in the database ofGenBank etc., probe set names or gene names on a gene chip and so forthare used.

In the present invention, the term “expression profile” meansinformation about change in expression level of each gene included inthe gene panel. In a preferred embodiment, the expression level ismeasured and recorded over time. In the expression profile, expressionlevel of a gene may be represented by an absolute value or relativevalue.

In a preferred embodiment of the gene panel of the present invention,genes are classified according to the expression levels at certain timesafter hepatectomy. For example, genes are classified into a group inwhich the expression levels markedly increase 1 hour and 6 hours afterhepatectomy (early stage of liver regeneration) (Groups 1 and 2), agroup in which the expression levels conversely decrease at this stage(Groups 6 and 7), a group in which the expression levels markedlyincrease 24 hours and 48 hours after hepatectomy (mid stage of liverregeneration) (Groups 3 and 4), a group in which the expression levelsconversely decrease at this stage (Groups 8 and 9), a group in which theexpression levels markedly increase 7 days after hepatectomy (laterstage of liver regeneration) (Group 5) and a group in which theexpression levels conversely decrease at this stage (Group 10). Theexpression “markedly” used herein means that the increase or decrease ofthe expression level is 3 times more compared with the level beforehepatectomy in the gene chip method, or that, in the case of theTaqman-PCR (SYBR Green) method, if a difference is observed in asignificance test, it is considered that there is a difference ofexpression.

In an embodiment of the gene panel of the present invention, thesegroups can be listed in the order of the length of the time for whichthe expression levels after the increase or decrease of expression aremaintained or for which the increase or decrease continues, startingwith the longest time as listed in Table 1.

Further, the gene panel preferably includes information about liverweight at a certain time after hepatectomy. For example, liver isextracted at an early stage of liver regeneration (1 hour and 6 hoursafter hepatectomy), mid stage of liver regeneration (24 hours and 48hours after hepatectomy) and later stage of liver regeneration (7 daysafter hepatectomy), and weight of each liver is measured.

The gene panel of the present invention may include sequence informationof PCR primers for analyzing expression level of each gene. Examples ofthe primers include primers comprising all or a part of theoligonucleotides of SEQ ID NOS: 1 to 127 and 135 to 192. Further, inaddition to the sequence information of the primers, nucleotide sequenceinformation of adaptors used in the ATAC-PCR method and/or the TaqmanPCR (SYBR Green) method may be included. SEQ ID NOS: 1 to 127 representprimers used in the ATAC-PCR method, and SEQ ID NOS: 135 to 192represent primers used in the Taqman PCR (SYBR Green) method. As for thenucleotide sequences of the adaptors, adaptors having the nucleotidesequence of SEQ ID NOS: 128 to 133 can be mentioned.

<2> Screening Method of the Present Invention

Based on the gene panel of the present invention, various screeningtests are enabled by constructing a system for quantitatively orsemi-quantitatively measuring expressions of genes included in the genepanel.

For example, screening for a drug involved in liver regeneration can beperformed by administering drugs to a model animal or liver tissue orcells and profiling expressions of genes constituting the gene panel ofthe present invention. That is, a drug that provides gene expressionprofiles similar to the expression profiles in the gene panel isconsidered to accelerate liver regeneration.

Further, a liver regeneration accelerating substance can also bescreened by screening for a drug that further increases or decreases theincreased or decreased expression levels of genes of the gene panel ofthe present invention. In this case, screening can be performed bypaying attention to expression changes of about 6 types of genes.Examples of the screening based on this conception will be shown in theexamples described later.

As the aforementioned 6 types of genes, for example, there can bementioned the genes of Nos. 5 (amino acid transporter system A (ATA2)),17 (high mobility group protein 2), 36 (nuclear RNA helicase), 46 (livernuclear protein p47), 67 (leukemia-associated cytosolic phosphoproteinstathmin) and 68 (deoxy-UTPase (dUTPase)) among the 137 types of genesin the gene panel shown in Table 1 ((1) to (11)).

Examples of the method for profiling gene expressions include the DNAmicroarray and DNA macroarray methods using a slide glass, nylonmembrane or the like on which fragments of genes constituting the genepanel are immobilized, the ATAC-PCR method, a method utilizing theTaqman probe, quantitative PCR utilizing SYBR Green and so forth. Theexpression profiling may be carried out by using a single kind of methodor two or more kinds of methods in combination.

Hereafter, a specific example of screening procedure will be described.

As primary screening, cultured hepatocytes are treated with drugs to bescreened or the drugs are administered to rats. After a certain time,RNAs are prepared from the cells or liver. The gene expression levelsare measured before and after the drug treatment, and drugs that provideexpression profiles similar to those in the gene panel are screened. Thegene expression patterns in hepatocytes after the drug treatment aregrouped, and drugs that provide grouping similar to the grouping in thegene panel are screened. The grouping is performed by classifying thegenes according to expression levels at a certain time in the samemanner as in the production of the gene panel.

As for culture conditions of hepatocytes, usual culture may be used, oraddition of substances that damage hepatocytes such as galactosamine,heavy metal ions or carbon tetrachloride to a medium and so forth may beused. It is expected that substances that accelerate liver regenerationor proliferation of cultured hepatocytes can be screened by culturingcells under the usual condition. Further, it is expected that substancesthat accelerate recovery of the liver function by liver regenerationafter cell damage can be screened by carrying out the culture in thepresence of a substance that damages hepatocytes.

As secondary screening, candidate drugs expected to exhibit action foraccelerating liver regeneration based on the results of the primaryscreening are administered to partially hepatectomized rats, and liveris extracted after certain intervals, for example, at an early stage ofliver regeneration (1 hour and 6 hours after hepatectomy), mid stage ofliver regeneration (24 hours and 48 hours after hepatectomy) and laterstage of liver regeneration (7 days after hepatectomy). The liverweights are measured, and RNAs are extracted from the livers to measurechanges in gene expression. The obtained data are compared with data ofliver weight changes and gene expression changes in partiallyhepatectomized rats not administered with the drugs to evaluate effectof the drugs on liver regeneration.

The aforementioned screening can also be performed by using laboratoryanimals other than rats. In this case, it is preferable to newly producea gene panel for homologues corresponding to the rat genes and performthe screening.

In addition to the screening of drugs that provide expression changessimilar to those in the gene panel, the following drug screening can beperformed by utilizing the gene panel. The genes that show the changesin the early stage of liver regeneration (for example, Groups 1, 2, 6and 7 mentioned above) are considered to be important for initiation ofliver regeneration. Therefore, it is expected that drugs effective forthe initiation of liver regeneration can be obtained by screening fordrugs that provide patterns similar to the patterns of these genes ofthese groups after the treatment with the drugs.

Further, the genes of which expression changes start in the mid stage ofliver regeneration (for example, those of Groups 3, 4, 8 and 9 mentionedabove) are considered to act on acceleration of proliferation ordifferentiation of hepatocytes after the initiation of liverregeneration. Therefore, it is expected that drugs that positively acton the acceleration of proliferation or differentiation of hepatocytescan be obtained by screening for drugs that provide patterns similar tothe patterns of these genes after the treatment with the drugs.

The genes of which expression changes start in the later stage of liverregeneration (for example, those of Groups 5 and 10 mentioned above) areconsidered to be important for termination of liver regeneration andnormal hepatocyte functions after the completion of regeneration.Therefore, it is expected that drugs that negatively act on theproliferation of hepatocytes or drugs effective for normal hepatocytefunctions after the completion of regeneration can be obtained byscreening for drugs that provide patterns similar to the patterns ofthese genes after the treatment with the drugs. The drugs thatnegatively act on the proliferation of hepatocytes can be used forprevention of excessive proliferation of hepatocytes, and possibility ofuse thereof as drugs for liver cancer or the like is expected.

As described above, the gene panel of the present invention isconsidered to be useful also for screening for drugs effective in eachstage of liver regeneration (for example, initiation, cellularproliferation, differentiation, termination of cellular proliferationetc.), and it is considered that more effective liver regenerationagents can also be created by using drugs obtained as a result of theabove screenings.

L-alanine is known to have liver regeneration effect. However, sinceL-alanine is easily metabolized and disappears in a living body,sufficient drug efficacy cannot be obtained. Accordingly, drugs arebeing required which have the same drug efficacy as that of L-alanineand are imparted with better characteristics such as prolonged effectobtained through improved pharmacokinetics. Further, there aresubstances such as crude drugs, of which drug efficacy is known butamount is insufficient for use as a drug since it is hard to securetheir raw material and they act as a mixture of two or more kinds ofcomponents. Therefore, substances that have liver regeneration actionand can be sufficiently supplied are being required. The presentinvention enables screening of substances useful as drugs with highsensitivity and high efficiency.

<3> Method for Evaluating Condition of Liver of the Present Invention

The gene panel of the present invention can be used for a system forevaluating whether diseased liver shows progression towards regenerationin liver diseases such as hepatitis and cirrhosis. Further, a drugtreatment suitable for each patient can be performed by examining thegene expression change pattern of each patient. For example, a series ofdrugs that specifically control certain genes (gene group) in the genepanel of the present invention in a positive or negative manner arescreened. Then, drugs necessary for liver regeneration can be selectedby examining expressions of the genes in the gene panel of each patientwith liver disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a structure of adaptor.

FIGS. 2 to 4 show relative expression levels of genes involved inL-alanine metabolism, of which expressions change during liverregeneration after administration of L-alanine (relative expressionlevels based on the expression level of β-actin, which is taken as1000).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained more specifically with referenceto the following examples.

EXAMPLE 1

<1> Preparation of Regenerating Liver

Ten week-old F344/DvCrj (Fischer) rats were etherized. The abdomen wasincised, and the left lobe and the intermediate lobe of the liver wereextruded from the incision. The liver was bound with sutures between theleft lobe and the intermediate lobe and between the right lobe and thecaudate lobe. The right lobe and the caudate lobe were left in the body,and the left lobe and the intermediate lobe were resected. Then, theincision was closed with a suture. As for the animals which wereexamined at 0 hour after the excision, suturing was not performed, andthe remaining liver was successively extracted. The incision was openedagain at 1, 6, 12, 24 and 168 hours after the hepatectomy to extract theremaining liver (regenerating liver). The weight of the remaining livers(right lobe and caudate lobe) was about 30% of the weight of the wholerat liver (right lobe, left lobe, intermediate lobe and caudate lobe).

Three liver regeneration model rats were prepared for each group, andthe remaining liver weight at the time of extraction was measured. Theweight was 2±0.3 g (1.6 g) 0 hour after the excision, 1.7±0.3 g (1.4 g)1 hour after the extraction, 2.1±0.5 g (2.1 g) 6 hours after theextraction, 2.5±0.5 g (3.0 g) 24 hours after the extraction, 2.5±0.5 g(2.0 g) 48 hours after the extraction, and 5.6±0.3 g (5.8 g) 7 daysafter the extraction (the parenthesized numerical values are weights ofthe regenerating livers used for the measurement using a gene chipdescribed later).

<2> Purification of Total RNA

In a volume of 10 ml of ISOGEN (Nippon Gene) was added to 1 g of ratliver, and the liver was homogenized. The obtained homogenate wascentrifuged to collect supernatant. This supernatant was added with 200μl of chloroform per 1 ml of the added ISOGEN and lightly stirred. Themixture was left at room temperature for 2 minutes and centrifuged at15000 rpm at 4° C. for 10 minutes, and the aqueous layer was transferredto a new centrifuge tube. This aqueous layer was added with anequivalent volume of 2-propanol, left at room temperature for 5 minutesand centrifuged at 15000 rpm at 4° C. for 15 minutes. The supernatantwas discarded, and the precipitated pellet was added with 70% ethanoland centrifuged at 15000 rpm at 4° C. for 15 minutes. The pellet wasrinsed by removing the 70% ethanol. The rinsed pellet was dried at roomtemperature for 5 minutes and added with DEPC (diethylpyrocarbonate)-treated water to dissolve the pellet. Purification of thetotal RNA fraction obtained as described above was confirmed by 1%agarose gel electrophoresis.

As described above, total RNA was purified from each of the remaininglivers (regenerating liver) at 0, 1, 6, 24, 48 and 168 hours after thepartial hepatectomy of rats, and changes in expression levels of variousgenes were examined by using GeneChip (Affymetrix) and ATAC-PCR.

<3> Gene Expression Analysis by GeneChip

Gene expressions were analyzed by using GeneChip according to theprotocol recommended by Affymetrix. The procedure is shown below.

(1) Probe Synthesis

(i) Synthesis of Double-Stranded cDNA

First, double-stranded cDNA was synthesized from the total RNA preparedin <2> by using SUPERSCRIPT Choice System produced by Gibco BRL. In anamount of 15 μg of total RNA and 100 pmol of T7-(dT)₂₄ primer weredissolved in DEPC-treated water to a volume of 11 μl. A reaction wasallowed at 70° C. for 10 minutes, and then the reaction mixture wascooled on ice, added with 4 μl of 5×1st strand cDNA buffer (Gibco BRL),2 μl of 0.1×DTT (dithiothreitol, Gibco BRL) and 1 μl of 10 mM dNTP mix(Gibco BRL) and kept warm at 42° C. for 2 minutes. The reaction mixturewas added with 2 μg of reverse transcriptase (Superscript II RT) andallowed to react at 42° C. for 1 hour.

The aforementioned reaction mixture was added with DEPC-treated water,30 μl of 5×2nd strand reaction buffer, 3 μl of 10 mM dNTP, 1 μl of 10U/μl DNA ligase, 4 μl of 10 U/μl DNA polymerase I and 2 U/μl of RNase Hand allowed to react at 16° C. for 2 hours. Then, the reaction mixturewas added with 2 μl of 5 U/μl T4 DNA polymerase and allowed to react at16° C. for 5 minutes. The reaction mixture was added with 10 μl of 0.5 MEDTA. To this reaction mixture, an equal volume of a solution(phenol:chloroform=1:1) was added, and these were mixed by verticallyshaking the tube containing them. This mixture was centrifuged at 15000rpm at 4° C. for 10 minutes, and the aqueous layer was transferred to anew centrifuge tube. This aqueous layer was added with 1/10 volume of 3M sodium acetate and 3-fold volume of 100% ethanol and mixed well. Themixture was left at −80° C. for 10 minutes and then centrifuged at 15000rpm at 4° C. for 10 minutes. The precipitated pellet was rinsed twicewith 70% ethanol, dried at room temperature for 5 minutes and then addedwith 12 μl of DEPC-treated water.

(ii) Synthesis of Biotin-Labeled cRNA Probe

Subsequently, a biotin-labeled cRNA probe was synthesized from thedouble-stranded cDNA synthesized as described above by using Bio ArrayHigh Yield RNA Transcript Labeling Kit produced by Enzo. In a volume of5 μl of double-stranded cDNA, 17 μl of DEPC-treated water, 4 μl of 10×HYbuffer, 4 μl of 10×biotin-labeled ribonucleotide, 4 μl of 10×DTT, 4 μlof 10×RNase inhibitor mix and 2 μl of 20×T7 RNA polymerase were mixedand allowed to react at 37° C. for 4 hours.

Then, unreacted biotin-labeled ribonucleotides were removed from thesolution of the biotin-labeled CRNA probe synthesized as described aboveby using RNeasy produced by Qiagen. The biotin-labeled cRNA probesolution was added with 160 μl of DEPC-treated water, mixed with 700 μlof RLT buffer, then added with 500 μl of 100% ethanol and mixed well. Ina volume of 700 μl each of this solution was added to an RNeasy minispin column and centrifuged at 8000 rpm for 15 seconds. The elutedsolution was added to the RNeasy mini spin column again and centrifugedat 8000 rpm for 15 seconds. Subsequently, 500 μl of RPE buffer was addedto the RNeasy mini spin column and centrifuged at 8000 rpm for 15seconds. In a volume of 500 μl of RPE buffer was added to the RNeasymini spin column again and centrifuged at 15000 rpm for 2 minutes.

The RNeasy mini spin column which was adsorbed with the biotin-labeledcRNA probe and washed as described above was transferred to a newcentrifuge tube. In a volume of 30 μl of DEPC-treated water was added tothe RNeasy mini spin column and left at room temperature for 1 minute.The mixture was centrifuged at 8000 rpm for 15 seconds to elute apurified biotin-labeled cRNA probe solution.

Subsequently, the purified biotin-labeled CRNA probe solution wasfragmented. The biotin-labe led CRNA probe solution and 5× fragmentationbuffer (⅕ volume of the final solution volume was added) were mixed toobtain a biotin-labeled cRNA probe concentration of 0.5 μg/μl andallowed to react at 94° C. for 35 minutes. The reaction mixture wassubjected to 1% agarose gel electrophoresis to confirm that the probehad been cut into fragments having a length of about 100 bases.

(2) Hybridization

The fragmented biotin-labeled cRNA probe was first evaluated by using atest chip (TEST2 Chip) to confirm that the probe had no problem, andthen the objective test of hybridization was performed. Rat chip sets(RG-U34A, RG-U34B, RG-U34C) were used for the objective test. These 3types of rat chip sets include 7000 types of known rat genes and 17000types of unknown genes in total. Hybridization was performed by thefollowing procedure for the both cases using the test chip and the ratchip sets.

In an amount of 60 μg of the fragmented biotin-labeled cRNA probe, 12 μlof control oligonucleotide B2 (5 nM), 12 μl 100×control cRNA cocktail,12 μl of herring sperm DNA (10 mg/ml), 12 μl of acetylated BSA (50mg/mg) and 600 μl of 2×MES hybridization buffer were added, and thetotal volume was adjusted to 1200 μl with DEPC-treated water (referredto as “hybridization cocktail” hereinafter). The hybridization cocktailwas heated at 99° C. for 5 minutes to cause heat denaturation, left at45° C. for 5 minutes and centrifuged at 15000 rpm at room temperaturefor 5 minutes. For hybridization, the supernatant was added in a volumeof 80 μl to the test chip (TEST2 Chip) or in a volume of 200 μl to eachof the rat chip sets (RG-U34A, RG-U34B, RG-U34C).

The GeneChip was returned to room temperature, and prehybridization wasperformed with 1×MES buffer (80 μl for the TEST2 chip and 200 μl for therat chip sets) at 60 rpm at 45° C. for 10 minutes. Subsequently, theprehybridization solution was removed, and the aforementionedheat-denatured hybridization cocktail was added. Hybridization wasallowed at 45° C. for 16 hours at 60 rpm.

(3) Washing, Staining and Scanning

The hybridization cocktail was removed from the GeneChip, and anon-stringent wash buffer was added to perform washing and staining byusing Fluidic Station (Affymetrix). The washing and staining wereperformed according to the Mini_euk1 protocol of Fluidic Station for theTEST2 Chip and according to the EukGE-WS2 protocol for the rat chipsets. After the completion of the washing and staining, the chip wasscanned with a scanner to obtain image data.

(4) Data Analysis

Data analysis of the hybridization was performed by using GeneChipAnalysis Suite. The expression level of each gene was represented by arelative value (average difference) based on the average value ofexpression levels of all the genes, which was taken as 100 (Table 1, (1)to (11)). In the table, the gene chip probe set names are identificationnumbers for management corresponding to the genes, which were designatedby Affymetrix. UniGene is a database of DNA sequences registered atGenBank compiled according to gene (transcription product) types andspecies.

The column of 0 hr in the table shows the gene expression levels in ratlivers examined at 0 hour after partial hepatectomy. Similarly, thecolumns of 1 hr, 6 hr, 24 hr, 48 hr and 7d show gene expression levelsin rat livers at 1, 6, 24, 48 hours and 7 days, respectively, afterpartial hepatectomy. The values in the columns of 0 hr/0 hr, 1 hr/0 hr,6 hr/0 hr, 24 hr/0 hr, 48 hr/0 hr and 7d/0 hr show relative values ofgene expression levels at 0, 1, 6, 24, 48 hours and 7 days after partialhepatectomy based on the gene expression level at 0 hour after partialhepatectomy. The second decimal place of a value was rounded off.Further, values of expression levels of 0 or less were assumed as avalue of 1 for convenience to obtain the expression ratio.

To produce a gene panel, known genes (genes of which sequence structuresincluding all ORF had been registered in the GenBank database excludingthose only of which partial sequences are known such as EST) were used.

For 77 types of known genes, expression levels in regenerating livermarkedly increased as compared with the expression levels in normalliver (increased about 3 times or more, shown in boldfaces in Table 1).For 60 types of known genes, expression levels in regenerating livermarkedly decreased as compared with the expression levels in normalliver (decreased to about ⅓ or less, shown in italics in Table 1). Amongthese, 7 types of known genes showed marked expression increases as wellas decreases during liver regeneration. That is, marked expressionchanges (increases or decrease of 3 times or more from the expressionsbefore hepatectomy) were observed during liver regeneration for 137types of known genes. TABLE 1 (1) Rat UniGene No. GeneChip probe set No.Gene  1 U17254_g_at Rn.10000 immediate early gene transcription factorNGFI-B  2 J05460_s_at Rn.10737 P-450 cholesterol 7-alpha-hydroxylase  3E01524cds_s_at Rn.11359 NADPH-cytochrome P450 redutase  4AF030091UTR#1_g_at Rn.12962 cyclin ania-6a  5 rc_AI171231_at Rn.16393amino acid transporter system A (ATA2)  6 rc_AI070318_at Rn.17145connective tissue growth factor  7 M58587_at Rn.1716 Interleukin 6receptor  8 AF036335_g_at Rn.1926 NonO/p54nrb homolog  9 D12769_g_atRn.19481 BTE binding protein (same as 105-E9, 126-A2) 10 rc_AI103604_atRn.19491 deubiquitinating enzyme Ubp45 (ubp45) 11 rc_AA900505_at Rn.2042rhoB 12 rc_AA849767_at Rn.20670 brain-enriched SH3-domain protein 13rc_AI137820_at Rn.22129 CYR61 14 rc_AA849718_g_at Rn.2648interferon-inducible protein 16 15 rc_AI101099_at Rn.2714Metallothionein2 16 M60921_at Rn.27923 B-cell translocation gene 2,anti-proliferative (PC3) 17 rc_AI008836_s_at Rn.2874 high mobility groupprotein 2 18 S74351_s_at Rn.31120 protein tyrosine phosphatase (dualspecificity phosphate) 19 D37920_at Rn.33239 squalene epoxidase 20M58634_at Rn.34026 Insulin-like growth factor binding protein 1 21rc_AI014163_at Rn.3723 interferon-related developmental regulator 1(PC4) 22 AF020618_g_at Rn.37483 progression elevated gene 3 protein 23rc_AA900348_s_at Rn.40138 tip associating protein (TAP) 24rc_AI072078_at Rn.42905 protein kinase KID2 (Kid2) 25 X54686cds_atRn.44317 pJunB 26 J03190_at Rn.6274 5-aminolevulinate synthase 27S77528cds_s_at Rn.6479 rNFIL-6 = C/EBP-related transcription factor(C/EBP- beta) 28 AF023087_s_at Rn.9096 nerve growth factor inducedfactor A (Early growth response 1) 29 L27843_s_at Rn.9459 tyrosinephosphatase (PRL-1) 30 L41275cds_s_at Rn.10089 p21 (WAF1) 31rc_AI236828_s_at Rn.10247 Signal transducer and activator oftranscription 3 32 X68101_at Rn.10431 trg 33 M33962_g_at Rn.11317Protein-tyrosine phosphatase 34 rc_AI234949_at Rn.12262 phocein protein35 S85184_at Rn.1294 Cyclic Protein-2 = cathepsin L proenzyme 36AF063447_at Rn.14550 nuclear RNA helicase 37 M12919mRNA#2_at Rn.1774Aldolase A, fructose-bisphosphate 38 L23148_g_at Rn.2113 Inhibitor ofDNA binding 1, helix-loop-helix protein (splice variation) 39 U21871_atRn.2143 outer mitochondrial membrane receptor rTOM20 40 X16038exon_s_atRn.25737 alkaline phosphatase 41 X62952_at Rn.2710 vimentin 42 M24067_atRn.29367 Plasminogen activator inhibitor 43 J02722cds_at Rn.3160 hemeoxygenase 44 X58828_at Rn.33497 PTP-S mRNA for protein-tyrosinephosphatase 45 D16309_at Rn.3483 cyclin D3 46 rc_AA892014_s_at Rn.3516liver nuclear protein p47 47 U33500_g_at Rn.37873 retinol dehydrogenasetype II 48 J00692_at Rn.39438 skeletal muscle alpha-actin 49U75404UTR#1_s_at Rn.42891 Ssecks 322 (PKC binding protein) 50 U42627_atRn.4313 dual-specificity protein tyrosine phosphatase (rVH6) 51rc_AA997933_at Rn.43930 Nopp140 associated protein (NAP65) 52M14369exon#2_at Rn.44576 K-kininogen, differential splicing leads to HMWKngk 53 Y14933mRNA_s_at Rn.48812 hepatocyte nuclear factor 6 beta 54rc_AA900769_s_at Rn.6321 smooth muscle alpha-actin 55 rc_AI146033_atRn.8459 small zinc finger-like protein (TIM9a) 56 rc_AI176376_s_atRn.8925 ATPase Na+/K+ transporting beta 1 polypeptide 57 rc_AI230712_atRn.950 Subtilisin - like endoprotease 58 AB000717exons#1-8_s_at Rn.9855non-hepatic-type S-adenosylmethionine synthetase 59 M24604_at Rn.223proliferating cell nuclear antigen (PCNA/cyclin) mRNA 60rc_AA860030_s_at Rn.2458 class I beta-tubulin 61 S80431_s_at Rn.25716delta 4-3-ketosteroid 5 beta-reductase 62 J03786_s_at Rn.2586 CytochromP450 15-beta gene 63 rc_AI639082_s_at Rn.33226 mini chromosomemaintenance deficient 6 64 rc_AA818524_at Rn.36115 hnRNP protein,partial 65 rc_AA998683_g_at Rn.3841 Rat heat shock protein (Hsp27) 66M33329_f_at Rn.40124 hydroxysteroid sulfotransferase a (STa) 67rc_AI231821_at Rn.555 Leukemia-associated cytosolic phosphoproteinstathmin 68 U64030_at Rn.6102 dUTPase 69 M22670cds_g_at Rn.780alpha-2-macroglobulin 70 D14014_at Rn.9471 cyclin D1 71 L15079mRNA_s_atRn.9679 P-glycoprotein 3/multidrug resistance 2 72 V01227_s_at Rn.3441alpha-tubulin 73 M37584_at Rn.3636 Rat histone (H2A.Z) 74 rc_AA946368_atRn.3790 FAT 75 rc_AI230970_f_at Rn.11229 Hemoglobin, alpha 1 76rc_AA925864_at Rn.32702 aldose-reductase-like protein MVDP/AKR1-B7 77D14989_f_at Rn.40365 hydroxysteroid sulfotransferase subunit 78rc_AI234295_at Rn.16933 type IIb sodium-phosphate transporter 79rc_AI234100_f_at Rn.2401 cysteine rich protein 80 M11794cds#2_f_atRn.2714 Metallothionein 1(same as 6-E2) 81 J05210_at Rn.29771 ATPcitrate lyase 82 AF089825_at Rn.30020 activin beta E 83 rc_AA943735_atRn.32110 glycine transporter 84 X05684_at Rn.48821 Pyruvate kinase,liver and RBC 85 rc_AI009273_s_at Rn.9486 fatty acid synthase 86M96601_at Rn.9968 taurine/beta-alanine transporter 87 L16995_atadd1(helix-loop-helix protein associated with adipocyte determinationand differentiation) 88 X91234_at Rn.10515 17-beta hydroxysteroiddehydrogenase type 2 89 L37333_s_at Rn.10992 glucose-6-phosphatase(G6Pase) 90 D43964_at Rn.11129 bile acid-Coenzyme A dehydrogenase: aminoacid n- acyltransferase 91 X74593_at Rn.11334 Sorbitol dehydrogenase 92rc_AA852046_s_at Rn.11350 VL30 element (same as 12-C10) 93rc_AA892553_at Rn.12592 signal transducer and activator of transcription1 (Stat1) 94 rc_AI113166_s_at Rn.15739 Apolipoprotein C-IV 95rc_AA818627_at Rn.16736 ISI1_RAT INSULIN-INDUCED PROTEIN 1 96AB016800_g_at Rn.228 7-dehydrocholesterol reductase 97 rc_AI233209_atRn.24561 CDK108 98 rc_AA893485_at Rn.31772 Glu-Pro dipepeptide repeatprotein 99 M95591_at Rn.3252 farnesyl diphosphate farnesyl transferase 1100  L25785_at Rn.3545 Transforming growth factor beta stimulated clone22 101  D17370_at Rn.3881 cystathionine gamma-lyase 102 AJ011656cds_s_at Rn.4513 claudin 3 103  rc_AI177004_s_at Rn.51063-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 104  rc_AI172293_atRn.7167 RANP-1 105  rc_AA957498_at Rn.8548 androgen-inducible aldehydereductase 106  rc_AI232087_at Rn.10417 (S)-2-hydroxy acid oxidase 107 M18363cds_s_at Rn.10870 Cytochrome P450, subfamily IIC (mephenytoin 4-hydroxylase) 108  J02585_at Rn.10982 liver stearyl-CoA desaturase 109 rc_AI044610_s_at Rn.11064 Dopa decarboxylase (aromatic L-amino aciddecarboxylase) 110  X06150cds_g_at Rn.11142 glycine methyltransferase111  J05031_g_at Rn.147 Isovaleryl Coenzyme A dehydrogenase 112 rc_AA925393_at Rn.15516 acetyl-coenzyme A carboxylase 113 rc_AA926200_at Rn.15681 aldehyde oxidase (female form) 114  AF037072_atRn.1647 carbonic anhydrase 3 115  rc_AA819899_at Rn.1829 transcobalaminII precursor (TCII) 116  M22359mRNA_s_at Rn.1911 plasma proteinaseinhibitor alpha-1-inhibitor III 117  K00996mRNA_s_at Rn.2287 cytochromep-450e (2b19(Cyp2b15)) 118  M59861_at Rn.2328 10-formyltetrahydrofolatedehydrogenase 119  rc_AI169656_at Rn.23348 organic anion transporter 3120  X14552_at Rn.33965 Caldesmon 1 121  Y07534cds_s_at Rn.34396 Serineprotease inhibitor (cytochrome P451c27/25) 122  J02827_g_at Rn.3489Branched chain alpha-ketoacid dehydrogenase subunit E1 alpha 123 K03249_at Rn.3671 peroxisomal enoyl-CoA-hydrotase-3-hydroxyacyl- CoAbifunctional enzyme 124  rc_AI639418_at Rn.42914 type I thyroxinedeiodinase 125  U88036_at Rn.5641 brain digoxin carrier protein 126 X65296cds_s_at Rn.6066 carboxylesterase (Es-HVEL) (cholesterol esterase)127  D10354_s_at Rn.6318 alanine aminotransferase 128  rc_AI144586_atRn.8415 evectin-1 (EVT1) 129  M84719_at Rn.867 flavin-containingmonooxygenase 1 (FMO-1) 130  K01934mRNA#2_at Rn.9854 Thyroid hormoneresponsive protein (spot14) 131  M77479_at Rn.9913 Solute carrier family10 (sodium/bile acid cotransporter family), member 1 132  J03863_atRn.9918 serine dehydratase (SDH2) 133  L22339_g_at Rn.9937N-hydroxy-2-acetylaminofluorene (ST1C1) 134  X79081mRNA_f_at p450CYP2C11gene for cytochrome 135  rc_AA945418_at Rn.23013 sterol12alpha-hydroxylase P450 136  rc_AI231076_at Rn.3572 IBP4_RATINSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 4 PRECURSOR 137  U10697_s_atRn.10050 kidney microsomal carboxylesterase Information about primersfor ATAC-PCR Ratio of expression level after hepatectomy to Used Ratioof expression level after expression level before hepatectomy (0 hr)(ATAC) restriction hepatectomy to expression Correlation enzyme levelbefore hepatectomy with (MboI unless GeneChip probe Gene expressionintensity (0 hr) (GeneChip) GeneChip SEQ ID otherwise No. set 0 hr 1 hr6 hr 24 hr 48 hr 7 d 0 hr 1 hr 6 hr 24 hr 48 hr 7 d Group 0 hr 1 hr 6 hr24 hr 48 hr 7 d data NO. specified)  1 U17254_g_at 26 765 112 6 60 32 129 4.3 0.2 2.3 1.2 1 1.0 14.9 1.3 1.3 3.2 0.8 0.985 1  2 J05460_s_at 1681042 1 180 157 636 1 6.2 0 1.1 0.9 3.8 1 2  3 E01524cds_s_at 111 530 596147 95 124 1 4.8 5.4 1.3 0.9 1.1 1 1.0 6.4 7.1 2.6 2.3 1.8 0.982 3  4AF030091UTR#1_g_at 39 461 533 171 275 275 1 12 14 4.4 7.1 7.1 1 4  5rc_AI171231_at 94 966 1330 92 129 218 1 10 14 1 1.4 2.3 1 1.0 11.2 13.13.5 1.2 1.8 0.975 5  6 rc_AI070318_at 53 202 437 118 99 88 1 3.8 8.2 2.21.9 1.7 1 1.0 4.3 4.1 8.4 3.0 1.3 0.225 6  7 M58587_at 86 343 416 69 94114 1 4 4.8 0.8 1.1 1.3 1 1.0 1.5 1.5 1.0 0.9 1.3 0.875 7  8AF036335_g_at 12 36 114 56 54 37 1 3 9.5 4.7 4.5 3.1 1 1.0 140.5 676.2261.9 258.2 311.3 0.958 8  9 D12769_g_at 189 713 482 118 220 259 1 3.82.6 0.6 1.2 1.4 1 1.0 2.2 1.3 0.8 0.4 1.5 0.814 9 10 rc_AI103604_at 166978 99 279 267 237 1 5.9 0.6 1.7 1.6 1.4 1 1.0 0.3 0.9 1.0 0.9 0.5−0.787 10 11 rc_AA900505_at 28 222 133 91 193 83 1 7.9 4.8 3.3 6.9 3 11.0 4.4 2.0 2.1 4.1 5.0 0.579 11 12 rc_AA849767_at 71 589 328 307 161145 1 8.3 4.6 4.3 2.3 2 1 12 Nla III 13 rc_AI137820_at 44 244 103 71 9158 1 5.5 2.3 1.6 2.1 1.3 1 13 14 rc_AA849718_g_at 162 705 612 586 558964 1 4.4 3.8 3.6 3.4 6 1 14 15 rc_AI101099_at 264 1421 1374 236 137 5021 5.4 5.2 0.9 0.5 1.9 1 15 16 M60921_at 155 547 566 257 221 270 1 3.53.7 1.7 1.4 1.7 1 1.0 18.4 16.8 3.5 4.0 3.8 0.984 16 17 rc_AI008836_s_at4 40 33 166 245 112 1 10 8.3 42 61 28 1 1.0 0.3 0.2 1.0 1.6 2.2 0.562 1718 S74351_s_at 159 667 165 69 74 15 1 4.2 1 0.4 0.5 0.1 1 1.0 5.8 1.10.3 1.0 3.8 0.730 18 19 D37920_at 17 57 124 36 28 97 1 3.4 7.3 2.1 1.65.7 1 1.0 3.7 4.4 4.4 4.0 3.8 0.516 19 20 M58634_at 100 986 924 37 1 2411 9.9 9.2 0.4 0 2.4 1 1.0 20.9 9.5 1.2 3.6 5.7 0.882 20 21rc_AI014163_at 10 297 143 27 49 50 1 30 14 2.7 4.9 5 1 1.0 1.7 1.0 0.70.6 0.3 0.818 21 22 AF020618_g_at 40 333 60 66 66 25 1 8.3 1.5 1.7 1.70.6 1 1.0 1.3 0.9 0.4 0.9 0.4 0.693 22 23 rc_AA900348_s_at 40 286 346274 243 238 1 7.2 8.7 6.9 6.1 6 1 1.0 5.4 3.4 2.3 5.5 4.9 0.567 23 24rc_AI072078_at 86 378 122 34 70 70 1 4.4 1.4 0.4 0.8 0.8 1 1.0 7.1 1.40.6 0.9 1.0 0.992 24 25 X54686cds_at 52 240 195 21 38 53 1 4.6 3.8 0.40.7 1 1 25 26 J03190_at 255 1051 870 377 315 189 1 4.1 3.4 1.5 1.2 0.7 11.0 5.6 2.4 1.2 2.2 0.9 0.868 26 27 S77528cds_s_at 143 498 364 181 208158 1 3.5 2.5 1.3 1.5 1.1 1 1.0 4.4 3.3 1.7 1.9 1.7 0.988 27 28AF023087_s_at 107 1362 961 187 254 616 1 13 9 1.7 2.4 5.8 1 1.0 1.8 1.81.5 2.4 2.4 0.273 28 29 L27843_s_at 226 1061 1033 459 482 324 1 4.7 4.62 2.1 1.4 1 1 10.4 10.5 6.1 3.5 26.8 0.023 29 30 L41275cds_s_at 44 18235 98 126 20 1 0.4 5.3 2.2 2.9 0.5 2 30 31 rc_AI236828_s_at 240 2981128 249 176 745 1 1.2 4.7 1 0.7 3.1 2 31 32 X68101_at 11 28 49 69 11755 1 2.5 4.5 6.3 11 5 2 32 33 M33962_g_at 128 166 471 154 139 138 1 1.33.7 1.2 1.1 1.1 2 33 34 rc_AI234949_at 42 44 322 79 85 33 1 1 7.7 1.9 20.8 2 1.0 1.7 2.7 1.9 0.5 0.6 0.751 34 35 S85184_at 92 221 393 144 132125 1 2.4 4.3 1.6 1.4 1.4 2 1.0 2.3 4.1 3.1 1.6 3.4 0.691 35 36AF063447_at 22 22 70 124 118 73 1 1 3.2 5.6 5.4 3.3 2 1.0 3.2 2.2 3.11.8 1.1 0.154 36 37 M12919mRNA#2_at 79 97 359 185 151 84 1 1.2 4.5 2.31.9 1.1 2 37 38 L23148_g_at 19 44 134 153 79 49 1 2.3 7.1 8.1 4.2 2.6 21.0 2.6 5.3 3.7 4.5 1.0 0.781 38 39 U21871_at 195 265 768 432 502 338 11.4 3.9 2.2 2.6 1.7 2 39 40 X16038exon_s_at 31 51 354 75 49 51 1 1.6 112.4 1.6 1.6 2 40 41 X62952_at 29 58 95 176 189 93 1 2 3.3 6.1 6.5 3.2 21.0 3.7 9.4 7.9 10.7 8.1 0.808 41 42 M24067_at 31 69 210 59 48 45 1 2.26.8 1.9 1.5 1.5 2 42 43 J02722cds_at 18 31 762 57 36 57 1 1.7 42 3.2 23.2 2 1.0 0.6 4.2 2.2 1.9 0.8 0.887 43 44 X58828_at 7 12 124 19 58 22 11.7 18 2.7 8.3 3.1 2 44 45 D16309_at 32 32 180 95 79 48 1 1 5.6 3 2.51.5 2 45 46 rc_AA892014_s_at 43 58 233 110 185 137 1 1.3 5.4 2.6 4.3 3.22 46 47 U33500_g_at 107 286 374 125 124 14 1 2.7 3.5 1.2 1.2 0.1 2 1.01.9 6.0 1.0 1.0 1.2 0.822 47 48 J00692_at 9 7 154 9 4 11 1 0.8 17 1 0.41.2 2 48 49 U75404UTR#1_s_at 23 31 141 36 38 28 1 1.3 6.1 1.6 1.7 1.2 249 Bfa I 50 U42627_at 25 51 103 63 104 45 1 2 4.1 2.5 4.2 1.8 2 1.0 2.02.8 1.7 3.8 6.1 0.219 50 51 rc_AA997933_at 73 52 592 214 178 163 1 0.78.1 2.9 2.4 2.2 2 51 52 M14369exon#2_at 39 40 385 17 34 105 1 1 9.9 0.40.9 2.7 2 53 Y14933mRNA_s_at 19 9 184 27 190 131 1 0.5 9.7 1.4 10 6.9 21 1.8 3.5 1.3 1.7 2.1 0.662 52 54 rc_AA900769_s_at 22 12 80 122 176 34 10.5 3.6 5.5 8 1.5 2 1.0 0.9 6.0 4.0 6.0 2.6 0.829 53 55 rc_AI146033_at159 151 701 281 281 180 1 0.9 4.4 1.8 1.8 1.1 2 54 Hpa II 56rc_AI176376_s_at 144 343 1274 335 283 147 1 2.4 8.8 2.3 2 1 2 55 57rc_AI230712_at 29 26 169 9 27 25 1 0.9 5.8 0.3 0.9 0.9 2 56 58AB000717exons#1-8_s_at 16 34 114 48 47 40 1 2.1 7.1 3 2.9 2.5 2 59M24604_at 118 184 257 820 442 224 1 1.6 2.2 6.9 3.7 1.9 3 60rc_AA860030_s_at 193 216 236 1119 699 463 1 1.1 1.2 5.8 3.6 2.4 3 57 61S80431_s_at 36 66 17 285 356 208 1 1.8 0.5 7.9 9.9 5.8 3 58 Bfa I 62J03786_s_at 84 183 162 416 555 170 1 2.2 1.9 5 6.6 2 3 1.0 3.5 5.8 6.612.3 7.2 0.841 59 63 rc_AI639082_s_at 41 36 27 191 100 76 1 0.9 0.7 4.72.4 1.9 3 1.0 0.7 1.1 0.7 3.0 2.7 0.027 60 64 rc_AA818524_at 21 26 57234 197 267 1 1.2 2.7 11 9.4 13 3 1.0 1.7 1.9 2.6 2.5 4.7 0.852 61 65rc_AA998683_g_at 73 181 65 394 259 37 1 2.5 0.9 5.4 3.5 0.5 3 1.0 0.90.7 1.7 3.9 1.2 0.516 62 66 M33329_f_at 180 406 134 633 633 896 1 2.30.7 3.5 3.5 5 3 1.0 1.5 0.4 0.9 1.3 2.6 0.795 63 67 rc_AI231821_at 37 5613 227 385 179 1 1.5 0.4 6.1 10 4.8 3 1.0 1.4 0.4 4.6 1.1 0.7 0.308 6468 U64030_at 29 38 28 207 233 97 1 1.3 1 7.1 8 3.3 3 1 0.4 0.3 0.9 0.60.4 0.270 65 69 M22670cds_g_at 64 71 105 210 54 47 1 1.1 1.6 3.3 0.8 0.73 66 Nla III 70 D14014_at 180 189 79 713 553 261 1 1.1 0.4 4 3.1 1.5 31.0 1.6 2.5 1.5 3.9 0.8 0.258 67 71 L15079mRNA_s_at 129 144 69 513 486184 1 1.1 0.5 4 3.8 1.4 3 1 2.3 1.1 3.5 1.9 1.4 0.740 68 72 V01227_s_at48 59 79 135 156 97 1 1.2 1.6 2.8 3.3 2 4 69 73 M37584_at 173 156 162447 693 450 1 0.9 0.9 2.6 4 2.6 4 70 Bfa I 74 rc_AA946368_at 34 45 79 86157 191 1 1.3 2.3 2.5 4.6 5.6 4 71 75 rc_AI230970_f_at 462 1181 856 12271031 1606 1 2.6 1.9 2.7 2.2 3.5 5 1.0 1.4 1.1 0.9 0.7 2.6 0.678 72 76rc_AA925864_at 22 4 50 55 64 358 1 0.2 2.3 2.5 2.9 16 5 1.0 1.4 1.5 2.73.3 2.7 0.458 73 77 D14989_f_at 147 296 101 414 334 586 1 2 0.7 2.8 2.34 5 74 Nla III 78 rc_AI234295_at 180 44 18 282 500 226 1 0.2 0.1 1.6 2.81.3 6 75 79 rc_AI234100_f_at 240 68 17 148 256 237 1 0.3 0.1 0.6 1.1 1 61.0 2.6 2.8 0.8 0.8 0.9 −0.883 76 80 M11794cds#2_f_at 1181 289 1 902 593912 1 0.2 0 0.8 0.5 0.8 6 1.0 5.3 19.8 2.6 1.2 3.9 −0.775 77 81J05210_at 533 124 112 61 110 446 1 0.2 0.2 0.1 0.2 0.8 6 1.0 0.9 0.1 0.40.1 0.1 0.314 78 82 AF089825_at 246 39 52 36 63 47 1 0.2 0.2 0.1 0.3 0.26 79 83 rc_AA943735_at 643 167 20 1 11 38 1 0.3 0 0 0 0.1 6 84 X05684_at214 35 107 24 22 100 1 0.2 0.5 0.1 0.1 0.5 6 80 85 rc_AI009273_s_at 1721369 513 313 262 2000 1 0.2 0.3 0.2 0.2 1.2 6 1.0 1.3 0.7 0.4 0.9 3.10.736 81 86 M96601_at 168 23 23 91 200 55 1 0.1 0.1 0.5 1.2 0.3 6 1 1.90.6 1.0 0.3 0.7 −0.459 82 87 L16995_at 386 111 51 25 174 217 1 0.3 0.10.1 0.5 0.6 6 83 88 X91234_at 464 887 1 1 22 406 1 1.9 0 0 0 0.9 7 1.02.5 0.1 0.1 0.0 0.1 0.892 84 89 L37333_s_at 674 592 102 229 162 577 10.9 0.2 0.3 0.2 0.9 7 1.0 2.0 0.4 0.3 0.4 0.4 0.609 85 90 D43964_at 966832 218 749 699 707 1 0.9 0.2 0.8 0.7 0.7 7 86 Bfa I 91 X74593_at 759688 176 428 462 526 1 0.9 0.2 0.6 0.6 0.7 7 1.0 0.7 0.9 0.3 1.8 2.60.013 87 92 rc_AA852046_s_at 1524 1401 336 1430 1187 1292 1 0.9 0.2 0.90.8 0.8 7 88 93 rc_AA892553_at 55 58 1 17 143 58 1 1.1 0 0.3 2.6 1.1 789 Nla III 94 rc_AI113166_s_at 1549 941 480 436 177 283 1 0.6 0.3 0.30.1 0.2 7 1.0 1.7 1.2 0.5 0.4 0.7 0.517 90 95 rc_AA818627_at 743 472 203688 565 252 1 0.6 0.3 0.9 0.8 0.3 7 1.0 0.8 0.5 0.9 0.4 0.4 0.708 91 96AB016800_g_at 340 175 55 279 214 319 1 0.5 0.2 0.8 0.6 0.9 7 1.0 1.1 0.61.6 1.6 3.6 0.529 92 97 rc_AI233209_at 138 142 43 33 82 15 1 1 0.3 0.20.6 0.1 7 93 98 rc_AA893485_at 179 159 12 59 117 110 1 0.9 0.1 0.3 0.70.6 7 94 99 M95591_at 155 102 1 16 77 235 1 0.7 0 0.1 0.5 1.5 7 95 100 L25785_at 801 559 112 344 264 356 1 0.7 0.1 0.4 0.3 0.4 7 96 101 D17370_at 742 559 182 861 844 1039 1 0.8 0.2 1.2 1.1 1.4 7 102 AJ011656cds_s_at 262 389 53 406 354 266 1 1.5 0.2 1.5 1.4 1 7 103 rc_AI177004_s_at 172 164 21 92 134 280 1 1 0.1 0.5 0.8 1.6 7 1.0 1.4 0.31.7 0.4 1.9 0.649 97 104  rc_AI172293_at 284 286 85 192 182 491 1 1 0.30.7 0.6 1.7 7 1.0 1.3 0.2 1.2 0.5 1.3 0.788 98 105  rc_AA957498_at 249154 1 65 72 46 1 0.6 0 0.3 0.3 0.2 7 99 106  rc_AI232087_at 1152 1002557 344 570 264 1 0.9 0.5 0.3 0.5 0.2 8 100 107  M18363cds_s_at 14471228 1113 358 599 342 1 0.8 0.8 0.2 0.4 0.2 8 1.0 1.4 1.0 0.1 0.2 1.60.326 101 108  J02585_at 1386 899 1001 165 357 1453 1 0.6 0.7 0.1 0.3 18 102 Nla III 109  rc_AI044610_s_at 482 447 294 100 389 357 1 0.9 0.60.2 0.8 0.7 8 1.0 1.3 0.9 0.1 1.4 1.3 0.828 103 110  X06150cds_g_at 16051313 1180 511 972 1203 1 0.8 0.7 0.3 0.6 0.7 8 1.0 1.4 2.9 0.8 2.1 1.80.079 104 111  J05031_g_at 470 428 366 115 179 233 1 0.9 0.8 0.2 0.4 0.58 1.0 1.6 1.1 0.6 1.0 1.5 0.499 105 112  rc_AA925393_at 966 386 1465 162311 860 1 0.4 1.5 0.2 0.3 0.9 8 106 113  rc_AA926200_at 1060 1588 602120 684 983 1 1.5 0.6 0.1 0.6 0.9 8 1.1 1.4 0.9 0.4 1.8 2.9 0.370 107114  AF037072_at 909 787 627 91 148 140 1 0.9 0.7 0.1 0.2 0.2 8 1.0 1.10.6 0.0 0.4 0.8 0.822 108 115  rc_AA819899_at 340 268 236 104 73 152 10.8 0.7 0.3 0.2 0.4 8 1.0 2.3 1.4 1.1 1.0 1.1 0.407 109 116 M22359mRNA_s_at 481 724 375 149 99 247 1 1.5 0.8 0.3 0.2 0.5 8 1.0 1.61.1 0.3 0.2 1.8 0.629 110 117  K00996mRNA_s_at 935 796 522 215 299 511 10.9 0.6 0.2 0.3 0.5 8 1.0 1.6 6.7 3.0 3.4 5.3 −0.433 111 118  M59861_at611 565 311 178 283 425 1 0.9 0.5 0.3 0.5 0.7 8 1.0 1.7 1.0 0.3 1.1 1.90.582 112 119  rc_AI169656_at 1437 1303 786 482 485 61 1 0.9 0.5 0.3 0.30 8 113 120  X14552_at 151 271 100 52 108 14 1 1.8 0.7 0.3 0.7 0.1 8 114121  Y07534cds_s_at 659 571 535 186 224 546 1 0.9 0.8 0.3 0.3 0.8 8 1.03.4 2.0 0.6 1.4 3.5 0.497 115 122  J02827_g_at 234 212 171 20 144 132 10.9 0.7 0.1 0.6 0.6 8 1.0 1.1 1.2 0.5 1.0 2.3 0.262 116 123  K03249_at242 295 142 40 137 187 1 1.2 0.6 0.2 0.6 0.8 8 117 124  rc_AI639418_at731 553 426 110 182 165 1 0.8 0.6 0.2 0.2 0.2 8 1.0 1.4 0.4 0.6 0.1 0.80.623 118 125  U88036_at 674 819 553 123 642 568 1 1.2 0.8 0.2 1 0.8 81.0 1.1 0.8 0.1 0.4 0.7 0.805 119 126  X65296cds_s_at 1046 1004 783 15874 216 1 1 0.7 0.2 0.1 0.2 8 120 127  D10354_s_at 640 497 366 103 274131 1 0.8 0.6 0.2 0.4 0.2 8 1 1.4 1.1 0.6 0.7 0.6 0.789 121 128 rc_AI144586_at 459 344 213 132 148 176 1 0.7 0.5 0.3 0.3 0.4 8 122 129 M84719_at 295 728 182 37 80 63 1 2.5 0.6 0.1 0.3 0.2 8 1.0 2.7 0.7 0.30.4 1.0 0.954 123 130  K01934mRNA#2_at 1125 982 837 124 212 1065 1 0.90.7 0.1 0.2 0.9 8 131  M77479_at 1124 1103 738 355 661 705 1 1 0.7 0.30.6 0.6 8 1.0 1.9 1.0 0.6 1.1 2.0 0.442 124 132  J03863_at 396 960 90719 197 594 1 2.4 2.3 0 0.5 1.5 8 1 5.5 4.7 0.5 0.9 1.2 0.902 125 133 L22339_g_at 1630 1305 1339 395 925 1263 1 0.8 0.8 0.2 0.6 0.8 8 1 1.20.8 0.1 0.3 0.2 0.709 126 134  X79081mRNA_f_at 588 587 401 53 74 63 1 10.7 0.1 0.1 0.1 8 135  rc_AA945418_at 3397 2633 2290 2194 699 1135 1 0.80.7 0.6 0.2 0.3 9 136  rc_AI231076_at 675 789 891 510 144 529 1 1.2 1.30.8 0.2 0.8 9 137  U10697_s_at 996 984 685 497 541 311 1 1 0.7 0.5 0.50.3 10 127 Correlation coefficient >= 0.7 34.000 45% Correlationcoefficient >= 0.5 50.000 67% Number of genes measured by 75.000ATAC-PCR<4> Gene Expression Analysis by ATAC-PCR

ATAC-PCR is a technique developed by Kato et al. (Kato, K et al., Nucl.Acids Res., 25, 4694-4696, 1997) and based on competitive RT-PCR usingfluorescent primers. Quantitative expression analysis of a large numberof genes can be conveniently carried out by ATAC-PCR. The experimentalprocedure of the ATAC-PCR method was according to the method of Kato etal. (Kato, K et al., Nucl. Acids Res., 25, 4694-4696, 1997).

First, double-stranded DNA was synthesized from cDNA synthesized byusing a 5′ biotinylated oligo dT primer and digested with a particularrestriction enzyme (example utilizing MboI is explained herein).Subsequently, an adaptor having the same end sequence as that of thesite digested with the restriction enzyme (6 types having differentlengths were prepared) and the double-stranded DNA digested with therestriction enzyme MboI were ligated with DNA ligase. Among the 6 typesof adaptors, 3 types were ligated to control cDNA (cDNA prepared fromrat liver before hepatectomy) and mixed at a ratio of 10:3:1. Theremaining 3 types of adaptors were ligated with cDNA prepared from therat liver at 1, 6, 24, 48 hours and 7 days after the hepatectomy.

The ligation reaction products were mixed, 3′ fragments of thedouble-stranded cDNA were recovered by using streptavidin-coated beads,and competitive RT-PCR was performed by using primers having a sequencecommon to all of the adaptors. The PCR products were analyzed by usingABI PRISM 3700 DNA Analyzer. This apparatus can separate fragments ateach length by capillary electrophoresis and detect fluorescenceintensity proportional to each expression level.

A calibration curve was prepared from fluorescence intensity of the PCRproducts obtained from the control, and gene expression change amountsin resected liver to be measured were calculated by using thecalibration curve.

The nucleotide sequences of the used adaptors are shown below. (Adaptor1, SEQ ID NO: 128) 5′ -GTACATATTGTCGTTAGAACGCG-3′3′ -CATGTATAACAGCAATCTTGCGCCTAG-5′ (Adaptor 2, SEQ ID NO: 129)5′ -GTACATATTGTCGTTAGAACGCGACT-3′ 3′ -CATGTATAACAGCAATCTTGCGCTGACTAG-5′(Adaptor 3, SEQ ID NO: 130) 5′ -GTACATATTGTCGTTAGAACGCGCATACT-3′3′ -CATGTATAACAGCAATCTTGCGCGTATGACTAG-5′ (Adaptor 4, SEQ ID NO: 131)5′ -GTACATATTGTCGTTAGAACGCGATCCATACT-3′3′ -CATGTATAACAGCAATCTTGCGCTAGGTATGACTAG 5′ (Adaptor 5, SEQ ID NO: 132)5′ -GTACATATTGTCGTTAGAACGCGTCAATCCATACT-3′3′ -CATGTATAACAGCAATCTTGCGCAGTTAGGTATGACTAG-5′ (Adaptor 6, SEQ ID NO:133) 5′ -GTACATATTGTCGTTAGAACGCGTACTCAATCCATACT-3′3′ -CATGTATAACAGCAATCTTGCGCATGAGTTAGGTATGACTAG-5′

As for the primer set used for the competitive PCR, the primer havingthe following nucleotide sequence was commonly used for the adaptor-sideof all genes, and the sequences shown in Table 1 and Sequence Listing(shown in a direction from 5′ to 3′) were used as gene-specific primers.(Adaptor-side primer) 5′ -GTACATATTGTCGTTAGAACGC-3′ (SEQ ID NO: 134)

For the genes of which expression changes were examined by ATAC-PCRusing restriction enzymes other than MboI, names of the used restrictionenzymes are shown in Table 1. Further, in such cases, adaptor sequenceshaving an end sequence different from that used for MboI were used. FIG.1 shows sequences of the different portions (boldfaces).

<5> Application of Gene Panel to Screening for Candidate LiverRegeneration Accelerating Substance

An experiment was performed to demonstrate that the gene panel of thepresent invention was effective for screening for liver regenerationaccelerating substances.

L-alanine is known to have effect of accelerating liver regeneration(Maezono K et al., Hepatology 24(5), 1996, Effect of Alanine onD-Galactosamine-Induced Acute Liver Failure in Rats; Japanese PatentLaid-open Publication No. 5-229940). Therefore, rats were subjected topartial hepatectomy and orally administered with L-alanine at 18 and 21hours after the hepatectomy, and then gene expressions at 24 hours afterthe hepatectomy were examined.

Five 6 week-old F344 male rats (120 g) were preliminarily fed for 6 daysby giving feed CRF-1 (Oriental Yeast) and water and then divided into 2groups (Group 1 and Group 2) each consisting of rats with the same bodyweight. At 24 hours before dissection, 70% partial hepatectomy wasperformed by the method by Higgins-Anderson (Higgins, G M and Anderson,R M, Arch. Pathol. 12, 186-191, 1931). On the day of autopsy, the ratswere starved for 6 hours before dissection. At 6 and 3 hours beforedissection, 2 g/10 ml/kg of L-alanine was orally administered as anaqueous solution containing 0.3% aqueous carboxymethylcellulose (CMC).

After the rats were killed by bleeding under anesthesia, the dissectionwas performed. Liver was extracted, weighed, cryopreserved, and thensubjected to the mRNA expression measurement by the ATAC-PCR method.Further, liver samples were prepared to measure the liver proliferatingcell nucleus antigen (PCNA) levels.

Gene expressions in rat liver in each group were measured by ATAC-PCR.The preparation of RNA and ATAC-PCR were performed as described above.In ATAC-PCR, among the genes in the gene panel, 117 types of whichexpression could be measured by ATAC-PCR using the restriction enzymeMboI were measured (those except for Nos. 12, 49, 52, 55, 58, 59, 61,69, 73, 77, 83, 90, 93, 101, 102, 108, 130, 134, 135 and 136 mentionedin Table 1).

Further, the livers of rats administered with L-alanine were stained byusing anti-PCNA antibodies (antibodies directed to proliferating cellnuclear antigen (PCNA), a replication factor that accelerates activityof DNA polymerase ä), and the PCNA labeling index was measured by themethod of Tanaka et al. (Tanaka et al., Byori to Rinsho, 9 (6): 791-798,1991).

The measurement results of the PCNA labeling index are shown in Table 2.Due to the L-alanine administration, the PCNA labeling index in thelivers elevated as compared with the livers of the non-administered rats(Table 2). In Table 2, Ala+represents the L-alanine-administered group,and Ala-represents the L-alanine non-administered group. Thus,hepatocytes proliferation accelerating effect due to the L-alanineadministration was observed. TABLE 2 PCNA PCNA positive NegativeLabeling cells cells Index (%) 1-1 4 570 0.7% 1-2 5 578 0.9% 1-3 2 6580.3% Mean 0.6% SD 0.3% 2-1 468 247 65.5% 2-2 426 240 64.0% Mean 64.7% SD1.1%1-1 to 1-3: Group 12-1, 2-2: Group 1

Further, the gene expressions in Groups 1 and 2 were measured byATAC-PCR for the genes of the gene panel and compared. The expressionlevels were obtained as relative values of Group 1 based on the valuesof Group 1 (Ala+/Ala− in Table 3). As a result, there were found 7 typesof genes which showed 3 times or more of expression difference betweenthe L-alanine-administered group and L-alanine non-administered group(Table 3). TABLE 3 Number Ala+, 24 hr/Ala−, 24 hr 5 4.8 17 5.5 36 4.6 464.4 67 4.2 68 3.5 117 3.2

Subsequently, to compare expression changes of the genes in the genepanel and gene expression changes due to the L-alanine administration,the ratios of expressions of the genes in Group 2 to expressions of thegenes in the gene panel at 0 hour after the hepatectomy were calculated.The results are shown in Table 4. In Table 4, 1 hr, 6 hr, 24 hr, 48 hrand 7d represent relative expression values in the remaining liversbased on the gene expression in the liver before the hepatectomy at 1,6, 24, 48 hours and 7 days after the hepatectomy, respectively. When theexpression values of Ala− at 24 hr and Ala+ at 24 hr are compared,marked differences (3 times or more) in the expression levels wereobserved in both of Groups 1 and 2 for 7 types (Nos. 5, 17, 36, 46, 67,68 and 117) among the genes in the gene panel (Table 4). Among these,the results of 5 types (Nos. 17, 36, 46, 67 and 68) corelated with theresults of the genes in the gene panel. That is, for the genes in thegene panel which showed expression increases from the levels before thehepatectomy at 24 hours, expressions exceeding the increased expressionswere observed due to the L-alanine administration. On the contrary, forthe genes in the gene panel which showed expression decreases from thelevels before hepatectomy at 24 hours, the expressions less than thedecreased expressions were observed due to the L-alanine administration.

For the remaining 2 types (Nos. 5 and 117) other than the above, thegenes of the gene panel did not show increased or decreased expressionchange due to the L-alanine administration at 24 hours. However, for No.5, expression increase was observed due to the L-alanine administrationat the maximum expression times (6 hr and 1 hr). As a result, it can beconstrued that expressions higher than the expressions of the genes inthe gene panel were maintained at 24 hours. On the other hand, since No.117 showed decreased change (decrease of expression) with the L-alanineadministration compared with that observed without L-alanineadministration at 24 hours, it is determined that it is not suitable foruse in screening for candidate liver regeneration acceleratingsubstance. TABLE 4 Ala− Ala+ Number 1 hr 6 hr 24 hr 48 hr 7 d 24 hr 510.3 14.1 1 1.4 2.3 4.8 17 10 8.3 41.5 61.3 28 228.3 36 1 3.2 5.6 5.43.3 25.8 46 1.3 5.4 2.6 4.3 3.2 11.4 67 1.5 0.4 6.1 10.4 4.8 25.6 68 1.31 7.1 8 3.3 24.9 117 0.9 0.6 0.2 0.3 0.5 0.6

Based on the above results, it can be considered that, in screeningutilizing the gene panel of the present invention, if a substanceprovides marked expression changes (3 times or more) of about 6 genesand the changes corelate to those in the gene panel (genes showingexpression increase in the gene panel show further expression increases,and genes showing expression decrease in the gene panel show furtherexpression decreases), such a substance can be selected as a candidateliver regeneration accelerating substance.

EXAMPLE 2 Expression Analysis of Genes Involved in L-alanine MetabolismDuring Liver Regeneration by Tagman PCR (SYBR Green)

In the same manner as in the above <1> and <2>, total RNA was purifiedfrom the remaining livers (regenerating livers) at 0, 1, 6, 24, 48 and168 hours after the partial hepatectomy of rats, and changes inexpression levels of various genes were examined by using the Taqman PCR(SYBR Green) method.

(1) Preparation of Template

The synthesis of template cDNA used for Taqman PCR (SYBR Green) wasperformed by using SuperScript First-Strand Synthesis System for RT-PCR(GIBCO BRL). In an amount of 500 ng of total RNA, 1 μl of 0.5 μg/μlOligo (dT)₁₂₋₁₈ and 1 μl of 10 mM dNTP mix were dissolved inDEPC-treated water to obtain a total volume of 10 μl. A reaction wasallowed at 65° C. for 5 minutes, and the reaction mixture was cooled onice, added and mixed with 2 μl of 10×RT buffer, 4 μl of 25 mM MgCl₂, 2μl of 0.1 M DTT and 1 μl of RNase Inhibitor, and kept warm at 42° C. for2 minutes. The reaction mixture was added with 1 μl (50 units) ofreverse transcriptase (Superscript II RT), and a reaction was allowed at42° C. for 50 minutes and further at 70° C. for 15 minutes.

(2) Selection of Candidate Gene and Design of Primer

The expression profiles of the genes involved in L-alanine metabolismduring liver regeneration were examined. To exhaustively extract ratgenes involved in L-alanine metabolism, the external database UniGene(http://www.ncbi.nlm.nih.gov/UniGene) was searched with keywords relatedto amino acid transporters, TCA cycle, respiratory chain and variouscarriers, and 59 types of genes of which sequences were known or forwhich primers could be designed were retrieved. Among these, 42 typeswere among the genes analyzed by using GeneChip in Example 1.

The primers used in Taqman PCR (SYBR Green) were designed by using theexternal database Primer3(http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The namesand Unigene Nos. of the genes and the nucleotide sequences of theirprimers are shown in Table 6. The table includes only the genes of whichexpression changes were observed as a result of the analysis by TaqmanPCR (SYBR Green).

(3) Taqman PCR (SYBR Green)

Taqman PCR (SYBR Green) was performed by the SYBR Green method(Schmittgen T D et al., Analytical Biochem., 285, 194-204, 2000).Specifically, the reaction was performed as follows. A reaction mixturehaving the composition shown in Table 5 was mixed in a PCR tube forTaqman, and PCR was performed by using ABI 7700 Prism Sequence Detector(ABI). The rection was performed with a cycle consisting of reactions at50° C. for 2 minutes and at 95° C. for 10 minutes followed by reactionsat 95° C. for 15 seconds and at 60° C. for 1 minute, which was repeated40 times. TABLE 5 Composition of PCR reaction mixture (per tube)Template cDNA (corresponding to 0.1 μl 2.5 ng of total RNA) 5 units/μlAmpliTaq Gold 0.05 μl dNTP mix (2.5 mM each) 0.8 μl 25 mM MgCl₂ 1.2 μl10 × SYBR buffer 1 μl Primer solution (forward) 1 μl Primer solution(reverse) 1 μl Distilled water Total: 10 μl(3) Data Analysis

All the Taqman PCR (SYBR Green) reactions were performed in duplicate,and the cycle threshold (CT) was calculated. The expression level atCT=30 was defined as 1, and a relative expression level was obtainedaccording to the following equation. “CT sample” represents CT of a genemeasured.Relative expression level=2^((30−CT sample))

Further, a relative value of the expression level of each gene wasobtained based on the relative expression level of β-actin, which wastaken as 1000. The results of the genes of which expressions changed inregenerating livers compared with those in normal liver are shown inTable 6. It is noted that, among the genes which showed expressionchange in regenerating livers in Example 1, expression changes were notreproduced for the genes of Nos. 81 (Rn.29771) and 131 (Rn.9913)mentioned in Table 1. TABLE 6 Primer Ratio of expression level aftersequence (SEQ hepatectomy to expression level before Unigene ID NO.)hepatectomy (0 hr) No. No. (Rn.) Gene 5′ end 3′ end 0 hr 1 hr 6 hr 24 hr48 hr 7 d 138 32067 Transporter protein; system N1 Na+ and H+-coupled135 136 1.00 0.96 0.60 0.60 0.37 0.86 glutamine transporter 139 53981Acetyl-CoA transporter 137 138 1.00 0.95 0.39 0.04 0.29 0.82 140 54434Organic cation transporter OCTN1 139 140 1.00 0.98 0.04 0.07 0.13 0.58141 14539 RAT BILE SALT EXPORT PUMP (ATP-BINDING CASSETTE, 141 142 1.000.95 0.55 0.34 0.58 0.79 SUB-FAMILY B 142 29977 MRP3 RAT CANALICULARMULTISPECIFIC ORGANIC ANION 143 144 1.00 0.70 1.25 1.30 3.78 0.70TRANSPORTER 2 143 10240 Solute carrier family 1, member 2 145 146 1.002.74 0.45 0.17 0.60 1.51 144 23204 Rattus norvegicus mRNA for LRp105,complete cds 147 148 1.00 1.62 0.88 0.24 0.32 1.01 145 11094 Pyruvatecarboxylase 149 150 1.00 1.14 0.40 0.11 0.36 0.78 146 11363 Pyruvatedehydrogenase kinase 2 subunit p45 (PDK2) 151 152 1.00 1.07 0.50 0.200.31 0.98 147 3766 Rat mitochondrial succinyl-CoA synthetase alphasubunit 153 154 1.00 1.03 0.74 0.57 0.98 0.78 (cytoplasmic precursor)mRNA, complete cds 148 3628 Glutamate oxaloacetate transaminase 2,mitochondrial 155 156 1.00 0.75 0.63 0.48 0.68 0.83 (aspartateaminotransferase 2) 149 6085 Solute carrier 16 (monocarboxylic acidtransporter), member 1 157 158 1.00 0.46 1.50 0.84 0.93 0.72 150 8368Solute carrier family 25 (mitochondrial carrier; citrate 159 160 1.000.55 0.40 0.57 0.60 0.85 transporter) member 1 151 5819Glutamic-oxaloacetic transaminase 1, soluble (aspartate 161 162 1.003.18 11.32 1.40 0.69 1.33 aminotransferase, cytosolic) see also D1Mgh12

EXAMPLE 3 Expression Analysis of Genes Involved in L-Alanine Metabolismby Tagman PCR (SYBR Green) with L-Alanine Administration During LiverRegeneration

Expression changes were examined with L-alanine administration duringliver regeneration regarding 59 types of rat genes involved in L-alaninemetabolism extracted in Example 2.

(1) Preparation of Regenerating Liver

Five 6 week-old F344 male rats (120 g) were fed for 6 days by givingfeed CRF-1 (Oriental Yeast) and water and then divided into 2 groups(Group 1 and Group 2) each consisting of rats with the same body weight.At 24 hours before dissection, 70% partial hepatectomy (left lobe andintermediate lobe) was performed by the method of Higgins-Anderson(Higgins, G M and Anderson, R M, Arch. Pathol. 12, 186-191, 1931). Onthe day of autopsy, the rats were starved for 6 hours before thedissection. At 18 and 21 hours after the partial hepatectomy, 2 g/10ml/kg BW of L-alanine was orally administered as an aqueous solutioncontaining 0.3% carboxymethylcellulose (Group 1), and only 0.3%carboxymethylcellulose was orally administered as a control (Group 2).

Dissection was performed at 24 hours after the partial hepatectomy.After the rats were killed by bleeding under anesthesia, livers wereextracted, weighed, cryopreserved and then subjected to the mRNAexpression measurement by the Taqman PCR (SYBR Green) method.

(2) Purification of Total RNA

Total RNA was purified from the remaining liver of 70% partiallyhepatectomized rats in the L-alanine administered group (Group 1) andthe control group (Group 2) in the same manner as in Example 1.

(3) Synthesis of Template

Templante cDNA used in Taqman PCR (SYBR Green) was synthesized in thesame manner as in Example 2.

(4) Desigan of Primers

Primers used in Taqman PCR (SYBR Green) were designed by using theexternal database Primer3(http://www-genome.wi.mit.edu/cgi-bin/primer/primer3 www.cgi). The namesand Unigene Nos. of the genes and the nucleotide sequences of theirprimers are shown in Table 7. The table includes only the genes forwhich expression changes were observed as a result of the analysis byTaqman PCR (SYBR Green).

(5) Taqman PCR (SYBR Green)

Taqman PCR (SYBR Green) was performed in the same manner as in Example2.

(6) Data Analysis

All the Taqman PCR (SYBR Green) reactions were performed in duplicate,and the cycle threshold (CT) was calculated. The expression level atCT=30 was defined as 1, and a relative expression level was obtainedaccording to the following equation.Relative expression level=2⁽30−CT sample)

Futher, a relative value of the expression level of each gene wasobtained based on the relative expression of β-Actin, which was taken as1000. The results of genes for which changes in expression level due tothe L-alanine administration were observed are shown in FIGS. 2 to 4.Among these genes, expression changes were also observed in 4 types ofgenes (Nos. 139, 140 and 151 mentioned in Table 6 and No. 166 mentionedin Table 7) among the genes of which expressions changed in regeneratingliver in Example 2.

INDUSTRIAL APPLICABILITY

The present invention provides expression information of genes involvedin liver regeneration. By utilizing this expression information, drugswhich accelerate liver regeneration or the like can be screened. TABLE 7Primer sequence UnigeneNO. (SEQ ID NO.) No. (Rn.) Gene 5′ end 3′ end 15248707 Cationic amino acid transporter-2A 163 164 153 29782 Fumaratehydratase 165 166 154 1093 Rattus norvegicus mRNA for NAD+specificisocitrate 167 168 dehydrogenase b-subunit, partial cds 155 2837 NAD(H)-specific isocitrate dehydrogenase gamma subunit 169 170 156 1504Rattus norvegicus cytosolic malate dehydrogenase (Mdh) 171 172 mRNA,complete cds 157 880 Cytochrome c oxidase subunit VIa (liver) 173 174158 1745 Cytochrome c oxidase subunit VIIa 3 175 176 159 2270 Rattusnorvegicus liver cytochrome c oxidase subunit VIII 177 178 (COX-VIII)mRNA, 3′ end of cds 160 19207 Rattus norvegicus mRNA for cytochrome Coxidase 179 180 assembly protein COX17, complete cds 161 10249Cytochrome b5, outer mitochondrial membrane isoform 181 182 162 80 ATPsynthase subunit d 183 184 163 3357 ATP synthase, H+ transporting,mitochondrial F0 complex, 185 186 subunit c (subunit 9), isoform 1 1649723 Rattus norvegicus (clone gamma-3) ATP synthase gamma- 187 188subunit (ATP5c) mRNA, 3′ end cds 165 853 2-oxoglutarate carrier 189 190166 32110 Rattus norvegicus glycine transporter mRNA, complete cds 191192

1. A gene panel comprising names of genes of which expression levelschange in hepatocytes during liver regeneration as compared with thosein a normal state and expression profiles of the genes.
 2. The genepanel according to claim 1, wherein the changes of the gene expressionlevels are changes in the expression levels in a model animal afterpartial hepatectomy as compared with the expression levels in a normalstate in the model animal.
 3. The gene panel according to claim 1 or 2,wherein the expression profiles include expression profiles over timeduring liver regeneration.
 4. The gene panel according to any one ofclaims 1 to 3, which includes sequence information of a group of PCRprimers for analyzing the expression profiles of the genes.
 5. The genepanel according to any one of claims 2 to 4, wherein the model animal isa rat.
 6. The gene panel according to any one of claims 1 to 5, whichincludes expression profiles of at least 6 types of genes among 166types of the genes represented by the numbers 1 to 166 in Tables 1, 6and
 7. 7. The gene panel according to claim 6, wherein the at least 6types of genes are selected from 151 types of the genes represented bynumbers 1 to 151 in Tables 1 and
 6. 8. The gene panel according to claim6, wherein the at least 6 types of genes are selected from 137 types ofthe genes represented by the numbers 1 to 137 in Tables 1 and
 6. 9. Thegene panel according to any one of claims 6 to 8, wherein the at least 6types of genes are the genes represented by the numbers 5, 17, 36, 46,67 and 68 in Table
 1. 10. A method for producing a gene panel comprisingexpression profiles of genes of which expression levels change inhepatocytes during liver regeneration as compared with those in a normalstate, which comprises the steps of: (a) measuring expression levels ofvarious genes in hepatocytes of a model animal in a normal state andexpression levels of the genes during liver regeneration; (b) comparingthe expression levels, respectively; and (c) identifying a group ofgenes of which expression levels change during liver regeneration andproducing expression profiles from information about gene names andchanges in the expression levels.
 11. The method according to claim 10,wherein, in the step (a), the expression levels of the gene are analyzedover time during liver regeneration.
 12. The method according to claim11, wherein a liver regeneration accelerating substance is administeredbefore, during or after liver regeneration.
 13. The method according toclaim 12, wherein the liver regeneration accelerating substance isL-alanine.
 14. The method according to any one of claims 10 to 13,wherein the gene expression levels are analyzed by one or more kinds ofmethods selected from the gene chip method, the ATAC-PCR method and theTaqman PCR (SYBR Green) method.
 15. The method according to claim 14,wherein the gene expression levels are analyzed by the gene chip methodand the ATAC-PCR method.
 16. The method according to claim 14, whereinthe gene expression levels are analyzed by the Taqman PCR (SYBR Green)method.
 17. A method for screening for a drug involved in liverregeneration, which comprises administering a drug to a model animal orliver tissue or cells and profiling expressions of genes constitutingthe gene panel according to claim
 1. 18. A method for evaluating acondition of liver, which comprises profiling expressions of genesconstituting the gene panel according to claim 1 for liver of a subject.19. A group of primers used in the method for producing a gene panelaccording to claim 10, the screening method according to claim 17 or theevaluation method according to claim 18, which comprises all or a partof the oligonucleotides of SEQ ID NOS: 1 to 127 and 135 to 192.